Development of immunoassay methods utilizing an antigen-antibody reaction is now being made on a global scale as an early detection method for new virus-based diseases such as AIDS and adult T-cell leukemia as well as various cancers. The methods are designed to detect an antibody or antigen itself utilizing the property of an antibody, which is prepared when virus or a like serving as an antigen has invaded into a living organism, to specifically react with the corresponding antigen (antigen-antibody reaction). As a micro-immunoassay method for this purpose, RIA (radioimmunoassay), EIA (enzyme-immunoassary), FIA (fluorescence-immunoassay), etc. have heretofore been used in practice. These methods use antigens or antibodies which are labeled with an isotope, an enzyme or a fluorescent substance in order to detect the presence of the corresponding antibodies or antigens, respectively, that react therewith.
Among them, RIA is to quantitatively determine the amount of the specimen which contributed to the antigen-antibody reaction by measuring the amount of the radio-activity of the isotope fixed for labeling. Up to date, only this method is capable of ultramicro measurement in the order of picogram. However, RIA needs special installment since it has to handle radioactive substances and there have heretofore been restrictions with respect to time and place for use and the like in view of the half-life period of the radioactive substances, disposal of wastes and the like. In addition, those methods which use enzymes or fluorescent substances are designed to confirm the presence of antigen-antibody reactions and therefore they remain semi-quantitative and the detectability limit is in the order of nanogram. Therefore, it has been a demand for an immunoassay method which has a detection sensitivity in the same order as that of RIA but is free of restrictions when in use.
Examples of thus-far published methods in which a laser beam is used to detect the presence of antigen-antibody reaction include a method designed for detecting liver cancer, in which micro-particles of a plastic are provided with an antibody to AFP (alpha-faeto protein) and the change in the mass as the result of agglomeration between the plastic particles due to an antigen-antibody reaction is monitored by the change in the scattering or transmission of a laser beam. Reportedly, the detection sensitivity of this method is 10.sup.-10 g/mL, which is one hundred times higher than conventional laser methods, but still one hundred times less sensitive as that of RIA. Since the method utilizes the change in Brownian movement of antigens and antibodies in an aqueous solution, it is necessary to precisely control the temperature of the aqueous solution containing a specimen upon measurement, and the method is defective in that it is susceptible to influences from outside such as ambient temperature and vibration.
Also, there is an essential limitation in the improvement of the detection sensitivity of the conventional laser beam scattering measurement and a large amount of specimen is required since only a part of the aqueous solution in which the specimen is dispersed is irradiated. One approach proposed for obviating this defect is to use as a vessel capillary tubes having the same diameter as that of a laser beam in the laser beam scattering measurement with a view to reducing time for measurement and minimizing the amount of a specimen used (U.S. Pat. No. 4,605,305). However, there is a problem in that the detection sensitivity is decreased due to disturbance of measurement by the scattered light from the walls of the capillary tubes.
Further, magnetic micro-particles are used in an attempt to facilitate the adjustment of a specimen. More particularly, magnetic micro-particles are used in the removal or separation of various components in liquid samples. For example, U.S. Pat. Nos. 4,018,886 and 3,970,518 disclose methods in which magnetic micro-particles are used to collect a specified protein, the protein is cut off from the magnetic micro-particles and the precipitate obtained is observed with eyes. Also, U.S. Pat. No. 3,933,997 discloses a method in which a radioactive label is concentrated onto a specimen using magnetic micro-particles. In addition, methods prevail in which magnetic micro-particles bearing a receptor capable of being specifically bound to a specimen, and a label substance are used to separate unused label substances (enzymes, radioactive substances, fluorescent substances, etc.) using magnetic force (WO86/04684 and EP-A-30087). However, although some improvement in the degree or accuracy of separation is observed, the overall degree of separation is unsatisfactory since measurement itself is made according to conventional method and is controlled by the accuracy of the method used.
Further, U.S. Pat. No. 4,219,335 uses the above-described magnetic micro-particles in order to magnetically check up the presence of a specified specimen in a body fluid. More particularly, a body fluid sample is affixed to the surface of the particles coated with a receptor reagent having a specific reactivity to the specimen, and upon contacting with the surface an immuno-reagent which has a reactivity with the receptor reagent or a complex of the receptor reagent and the specimen, a substance labeled with a reactance label such as micro-particles of a magnetic substance is used as an immuno-reagent, which is applied to the surface of a suspension of the specimen, and then, the change in the electrical reactance such as dielectric constant, conductivity or magnetic permeability of the surface is measured after removing unused immuno-reagent from the surface. This method, in which measurement is made in order to directly check the presence or abundance of labeled micro-particles, is different in its principle from methods in which a laser beam is radiated into a measurement system including an immunocomplex labeled with micro-particles of the magnetic substance and corresponding outgoing light is optically measured.
Furthermore, generally when preparing an assay sample for RIA, EIA, FIA, as well as for the laser magnetic immunoassay described in this document, it is necessary to separate the labeled specimen from the unused labeling reagent. For example, with the ELISA method, one variation of the EIA technique, a standard antigen material is deposited on micro-plates. After incubating with the specimen solution, these micro-plates must then be washed in order to remove remaining unreacted specimen material. The specimen material now fixed to the micro-plates is then reacted with an enzyme labeling material and again, the micro-plates must be washed in order to remove unused labeling reagent, ordinarily five or six times. If this washing process is abbreviated, labeling reagent is likely to remain and the assay results will thereby be adversely effected. Thus, a one step process to remove unreacted reagent would be highly desirable.
A further limitation of the above described ELISA method is the fact the antigen-antibody complex forming process for fixing specimen to the micro-plates is limited to the surface of the micro-plates. Because an exceedingly minute quantity of the specimen is present, this reaction step requires a significant incubation period in order to insure complete reaction. For this reason, a means to increase surface area for reaction would be of of great utility in expediting the assay procedure.
It is found that the shape of the analyte solution surface presents a serious barrier to effective assaying. Namely, the above said EIA method etc., when using the heretofore type of microplate, the said test vessel has a hydrophobic nature and since it is difficult to wet the water surface shape is not ideal. Therefore, when using the existing test vessel, since the water surface slants, the reflection direction and amount of reflection of the incident laser beam cannot be determined. Accordingly, it is necessary to revise the laser beam axis and control the position of the light receiver that detects the signal from the specimen at every time a measurement is made.
In this way, and as in the parent application, a high-sensitivity laser magnetic sample vessel is provided in the present invention to make possible the yield of highly reproducible results. In the interference system of the present invention, in the light-scattering method, the light-absorption method, and the light-diffraction method, the test vessels of the present invention are always of identical shape when holding the sample, intensity of the light radiated from the sample is constant, and therefore the reproducibility of the measurements of the light scattered, the light absorbed, and the angle of diffraction, are assured.
In the following sections, embodiments of the present invention will be explained with reference to the illustrations; these embodiments do not limit the scope of the invention, but rather are examples, the invention being limited only by the claims. Further, the inventors have published an invention P.No. 62-184902 (laser magnetic immunoassay method and measurement device) to measure by the interference method but, from the results of research on the mechanism of the occurrence of interference it was understood that the water surface was convex in the down direction, i.e., indispensable for the formation of the meniscus. Also it was found that in the scatter, permeability, and diffraction methods, the water surface shape had a significant effect on the repeatability of the data.
The present inventors have previously investigated methods for detecting magnetic-labeled specimen using a magnetic head with high sensitivity and applied Japanese Patent Application No. Sho-62-104066 entitled "Detector for magnetic substances" and Japanese Patent Application No. Sho-62-137988 entitled "Magnetic immunoassay and apparatus therefor". However, the detection sensitivity thereof remains in the order of 1.times.10.sup.-9 g/mL, which is by a thousands times less sensitive as that of the laser magnetic immunoassay according to the present invention even when these methods of improving the detection sensitivity invented by the present inventors are applied.